During the previous 5 years of this project, we have defined one receptor-counter receptor pair that[unreadable] removes washed platelets, chilled for 2-4h, from the circulation. We proposed that chilling causes the[unreadable] GPIb/V/IX receptor complex (vWfR) to cluster on the surface of chilled platelets bringing exposed beta-N-acetyl[unreadable] glucosamine (beta-GlcNAc) residues on N-linked glycans of the GP1b-alpha subunit together, which[unreadable] leads to their recognition by the lectin domain of alphaM-beta2 receptors on phagocytes in the liver. Coverage[unreadable] of exposed beta-GlcNAc on the GP1b-alpha chain by galactose (galactosylation) rescues the loss of[unreadable] circulation in mice of platelets chilled under these conditions. However, storage of platelets in plasma[unreadable] in the cold for 48 h induces further changes that lead to a loss of circulation not rescued by[unreadable] galactosylation. We now postulate that long-term chilling (equal to or greater than 48h) of platelets in plasma leads to a[unreadable] "hyperclustering" of the vWFR. This increases the density of galactose residues in clusters on[unreadable] galactosylated platelets such that they reach a critical density that now results in clearance by[unreadable] hepatocytes and/or macrophages using their Asialoglycoprotein receptor (ASGPR), which recognizes[unreadable] exposed galactose. GP1b-alpha is linked to the underlying actin skeleton by filamin A (FLNa). We[unreadable] previously showed that chilling induces platelets to remodel their cytoskeleton and assemble actin.[unreadable] Aim 1 will determine if GP1b-alpha is involved in the removal of platelets stored in plasma for 48h and if the[unreadable] long-term changes are due to the binding of plasma components to platelets. If GP1b-alpha is central to the[unreadable] changes, we will investigate the role of FLNa and gelsolin as well as the underlying actin-connection in[unreadable] vWfR clustering. A pure population of FLNa-null platelets using Ore driven by the hematopoetic[unreadable] specific promoter GATA1 in mice using our conditional loxP allele of FLNa has been established. We[unreadable] will determine if platelets lacking FLNa or gelsolin circulate in wild type (WT) mice. We postulate that[unreadable] FLNa null platelets will be cleared. These results will therefore get at the mechanism, and importance,[unreadable] of clustering in platelet survival and in vivo function (in collaboration with Project 1- Dr. Wagner). If[unreadable] platelets lacking FLNa circulate, we will determine if they fail to cluster the vWf receptor in the cold[unreadable] following short-term storage in buffer or long-term storage in plasma. We will also investigate if[unreadable] prolonged cold platelet storage dissociates the GP1b-alpha-FLNa/b complex. Aim 2 will determine: (1) if[unreadable] sialylation facilitates the survival of refrigerated (> 48 hrs) galactosylated platelets; (2) identify platelet[unreadable] acceptor proteins for UDP-galactose and CMP-sialic acid; (3) determine which organ/cells clear[unreadable] galactosylated and/or sialylated platelets (in collaboration with Project 3 - Dr. von Andrian); (4)[unreadable] evaluate in vivo function of modified and refrigerated platelets (in collaboration with Project 1 - Dr.[unreadable] Wagner); and (5) establish a humanized adaptive immune system in mice that will allow us to assess[unreadable] the effects of platelet modification on refrigerated human platelet survival and function in vivo (in[unreadable] collaboration with Projects 1 and 3 - Drs. Wagner and von Andrian). Studies in this area have been[unreadable] limited by a lack of good animal models that evaluate both human platelet circulation and function[unreadable] following transfusion. Aim 3, if necessary, will identify GP1b-alpha independent changes in murine platelets[unreadable] refrigerated (> 48 hrs) in plasma under blood bank conditions that target them for removal and identify[unreadable] the phagocytic receptor that mediates the removal. The overall goal of this work is to identify new[unreadable] targets that can be modulated to prevent cold-induced platelet clearance and develop methodology to[unreadable] block them.